I'm trying to construct a protein ladder, which are markers used to indicate the molecular weight of proteins when running a SDS-PAGE. I have conjugated several different molecular weight proteins with dyes and ran them on a 12% gel. There is no problem with the size when I loaded each protein into a separate well, but when I combined all of the proteins together into 1 well, the lower molecular weight proteins tend to widen out. May someone please explain the reasoning behind this phenomenon or how I can prevent this from happening? I've attached my gel below. I ran the gel at 150V for 10 mins, followed by 200V for 36 mins.

But the buffer in the new ladder should be the same as the others. The ladder is basically combining 2ul of each of the 9 dye-conjugated proteins (~18ul total). The dye-conjugated protein that I loaded separately and combined in the ladder are from the same solution. In addition, I loaded 1X loading buffer dye in the empty wells. Any ideas? Also, I'm afraid that the bands will not be as prominent if I dilute it further.